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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Curr Opin Chem Biol. 2021 Jun 3;64:57–66. doi: 10.1016/j.cbpa.2021.04.011

Figure 2.

Figure 2

In vitro probe applications to study conformational dynamics and aggregation of amyloid proteins. (Top Left) Combined PTM and FRET pair labeling through SPPS or chemoenzymatic synthesis, Uaa mutagenesis and NCL to give homogenous αS pY39 for smFRET studies. (Top Right) Three color smFRET in αS, which was site-specifically labeled with three distinct dyes all via cysteine-maleimide chemistry, by combining thiazolidine capture of the N-terminal cysteine in the C-terminal fragment with dextran-aldehyde resin and NCL. (Bottom) αS labeled with ESIPT probe 7FME that populates excited states NH*, N*, and T* is used to monitor the levels of monomers (M), intermediates (I), and fibrils (F) based on contributions to the spectra indicated by the fluorophore colors. Spectra at different time points and the derived speciation of αS are shown for an aggregation reaction. (Top Left) Adapted from ref. [54], Copyright 2020, the American Chemical Society. (Top Right) Adapted from ref. [58], Copyright 2018, Cell Press. (Bottom) Adapted from ref. [60], Copyright 2018, Springer Nature.